Embryonic stem cells (ESC) hold the promise to revolutionize reparative medicine through the development of stem cell-based therapies. This potential centers on the intrinsic abilities of proliferating ES cells to self-renew and to mature into essentially any cell type (pluripotency). Transplanting stem cells into damaged myocardium is emerging as a novel means for both acute repair and treatment of end-stage heart failure. The crux for therapeutic success will lie in being able to identify and manipulate proliferating ESCs to differentiate specifically into cardiac muscle upon demand. This proposal uses a series of synergistic and sophisticated proteomic technologies to address discrete biological and clinical questions with respect to ESC. In this discovery based grant, we focus on 4 distinctive sub-proteomes and include both downstream validation and functional analysis in order to develop new tools and provide insight into the regulation of ESC. Specific aim 1 focuses on the identification of cell surface proteins, in particular the N-linked glycoproteome to create specific biomarker panel for the identification and monitoring of various stages of differentiation. Furthermore, we link the cell surface receptor identification with the secreted regulatory factors (e.g. paracrine factors) found during the same early stages of differentiation. Specific aim 2 focuses on differential phospho-proteome analysis of ES cells over the initial 24 hours following differentiation initiated by serum starvation. This is the same time period in which the transcription factor, B-myb, is phosphorylated. B-myb is potentially one of the earliest triggers of differentiation. Specific aim 3, investigates B-myb with respect to its regulation by phosphorylation, alterations in this protein complex and the functional consequences on the early stemness proteins (eg. Oct 3 and Nanog). Understanding the complex and dynamic inter-relationships within the ESC proteome is of basic biological interest, and has major clinical implications.